SUMMARY ? Project 3 West Nile virus (WNV) is a continuing and expanding public health threat. Over the past 10 years, WNV has become the leading cause of arboviral encephalitis in humans in the U.S., as well as in some other parts of the world. In 2015, all 48 states and the District of Columbia reported WNV infections in people, birds, or mosquitoes. Of the 2,060 human cases reported in 2015, 66% were classified as neuroinvasive disease. The majority of human WNV infections are asymptomatic and unreported but 20-30% of infected humans have a mild illness. Approximately 1%, mostly adults, develop neuroinvasive disease and more than 10,000 people in the U.S. are currently living with residual neurocognitive impairments and/or functional sequelae. The majority of studies on WNV infections in brain cells have been done in mice due to the limited availability of primary human brain cells for research. Recent advances in targeted differentiation of induced human pluripotent stem cells (iPSCs) into different types of neural cells provide a consistent and renewable resource of human brain cells for rigorous and reproducible research. Additional advances in 3D human brain organoid technology including a recently developed miniaturized spinning bioreactor (Project 1) provide novel models for research on neurotropic viruses. Under Aim 1 in Project 3, we will first characterize neurovirulent and avirulent WNV infections in various 2D human brain cell cultures and 3D brain organoid models developed by Project 1. In Aim 2, we propose to utilize neural 2D cultures, 3D organoids and co-culture systems (Project 1 and Scientific Core) to investigate host cell responses to WNV infection and virus-induced pathogenesis in brain cells. The same cells types/models and experimental conditions will be used for WNV in this project as for ZIKV in Project 2. This will allow us to generate directly comparable data on two human neurotropic flaviviruses with different clinical outcomes in human brain cells from the earliest stages of progenitor proliferation through the formation of organoids containing post-mitotic neurons. In Aim 3, we will validate the utility of the 2D and 3D cell models as anti-WNV drug screening tools by testing the effects of candidate drugs with confirmed anti-ZIKV or anti- apoptotic activity. We will also reconstitute a potent, natural, antiviral phenotype found in mice that specifically targets members of the genus Flavivirus in human brain cells to investigate viral product clearance and possible long-term functional consequences in ?cured? neurons.